M. Coppola

Tumor induction and life shortening in BC3F1 female mice at low doses of fast neutrons and X rays

Extension of previous investigations at this laboratory regarding life shortening and tumor induction in the mouse has provided more complete dose-response information in the low dose region of X rays and neutrons. A complete observation of survival and late pathology has been carried out on over 2000 BC3F1 female mice irradiated with single doses of 1.5 MeV neutrons (0.5, 1, 2, 4, 8, 16 cGy) and, for comparison, of X rays (4, 8, 16, 32, 64, 128, 256 cGy). Data analysis has shown that a significant life shortening is observable only for individual neutron doses not lower than 8 cGy. Nevertheless, assuming a linear nonthreshold form for the overall dose-effect relationships of both radiation qualities, an RBE value of 12.3 is obtained for the 1.5 MeV neutrons. The induction of solid tumors by neutrons becomes statistically significant at individual doses from 8 cGy and by X rays for doses larger than 1 Gy. Linear dependence on neutron dose appears adequate to interpret the data at low doses. A separate analysis of ovarian tumor induction substantiates the hypothesis of a threshold dose for the X rays, while this is not strictly needed to interpret the neutron data. A trend analysis conducted on the neoplasm incidence confirms the above findings. Death rates have been analyzed, and a general agreement between the shift to earlier times of these curves and tumor induction was found.

Influence of age on life shortening and tumor induction after x-ray and neutron irradiation

The main object of this study is to investigate the role of age on the susceptibility to radiation carcinogenesis and life shortening for different qualities of radiation. Over the last few years, a line of research at the Laboratory of Pathology, C.R.E. Casaccia, has been set up to study the effects of exposure to neutron irradiation, including observations on late effects (both neoplastic and nonneoplastic) as a function of radiation dose and of age at irradiation. Graded single doses of X rays or attenuated fission neutrons have been given to male BC3F1 mice 3 and 19 months old and to animals in utero at 17 days postcoitum. The analysis of data from over 3000 mice indicates that irradiation at 3 months of age causes life shortening which is associated with the incidence and rate of radiation-induced neoplasms. Prenatal irradiation or irradiation at 19 months of age does not show a clearly measurable life shortening for both X-ray and neutron exposures. However, significantly higher incidence and rate of solid tumors and reticulum cell sarcomas were observed. In general the data confirm the higher biological effectiveness of neutrons compared with X rays. The estimates of neutron relative biological effectiveness for different end points were found to be in the range of 3 to 18 and their variation was closely dose dependent.

Age-related susceptibility of mouse liver to induction of tumors by neutrons.

The induction of liver tumors has been studied in BC3F1 male mice after acute whole-body irradiation with fission neutrons and X rays, given at different ages. In prenatally irradiated mice, a small effect is seen after doses of 0.3 to 2.1 Gy of X rays, and a more pronounced effect is found after neutron doses of 0.09 to 0.62 Gy. At 3 months of age the animals show a higher incidence after X-ray doses from 2 Gy, and for neutrons from 0.17 Gy. At 19 months of age, liver tumors are rarely induced by either type of radiation. These findings are confirmed by the statistical analysis of trend. The possibility of deriving dose-response relationships for liver tumors was also investigated. In the dose ranges where the risk appears to increase as a function of the increase in dose, the data points for neutrons were well fitted by a linear expression. A pure quadratic relationship best fitted the X-ray data. Using these expressions, the RBE for neutrons was 28 at 0.09 Gy for prenatal irradiation and 13 at 0.17 Gy for irradiation at 3 months. This suggests the existence of a risk of developing liver tumors after exposure to radiation, and fetal liver seems to be particularly sensitive to neoplastic transformation. This risk may be negligible at low doses (less than 1 Gy) of low-LET radiation, or with exposure at older ages.

The dose-response relationships for myeloid leukemia and malignant lymphoma in BC3F1 mice

The present analysis of data on the induction of lymphoma and myeloid leukemia in BC3F1 mice has indicated some new and interesting aspects regarding the shapes of the dose-effect curves. The incidence data can be interpreted by radiobiological models of the induction process coupled with cell inactivation. In particular, for malignant lymphoma the dose-response curve after X rays can be described assuming a quadratic model corrected for cell inactivation, while the incidence data after fission neutrons are best fitted by a linear model which also allows for cell inactivation. Myeloid leukemia has also been induced in BC3F1 mice. The bell-shaped dose-response curves observed after irradiation with either X rays or neutrons are explained by assuming simultaneous initial transforming events and cell inactivation with the data for cell inactivation at higher doses being in agreement with data reported for other strains of mice. A value for relative biological effectiveness of 4 is obtained at the lowest neutron dose used. The value of the inactivation parameters can be compared with those of the cell inactivation probability per unit dose for the bone marrow hematopoietic stem cells, which are believed to be the target cells for these tumors.

The influence of sex on life shortening and tumor induction in CBA/Cne mice exposed to X rays or fission neutrons.

An experimental study of male and female CBA/Cne mice was set up at Casaccia primarily to investigate the influence of sex on long-term survival and tumor induction after exposure to high- and low-LET radiation. Mice were whole-body-irradiated at 3 months of age with fission-neutron doses of 0.1, 0.2, 0.4, 0.8, 1.2 and 1.8 Gy at the RSV-TAPIRO reactor (mean neutron energy 0.4 MeV, in terms of kerma, y(D) = 51.5 keV/micron), or with 250 kVp X-ray doses of 1, 3, 5 and 7 Gy. Control and irradiated animals were then followed for their entire life span. As a general finding, male CBA/Cne mice appear more susceptible to tumorigenesis than females. In particular, the incidences of induced acute myeloid leukemia and malignant lymphomas are significant only in male mice. Benign and malignant solid tumors of many types are observed in mice of both sexes, the most frequent being in the lung, liver and ovary. However, evidence for a radiation response is limited to the case of Harderian gland neoplasms. In addition, a comparison of the observed frequency of all irradiated compared to unirradiated animals bearing solid tumors shows that the total tumor occurrence is not altered markedly by radiation exposure. A decrease in survival time is observed for both sexes and radiation types and correlates well with increasing dose. Moreover, both sex and radiation quality appear to influence the life shortening. A similar dose dependence of survival time is found when tumor-free animals alone are considered, suggesting a non-specific component of life-shortening.

Sensitivity of C3H10T1/2 cells to radiation-induced killing and neoplastic transformation as a function of cell cycle

Cell-age sensitivity to both cell killing and neoplastic transformation induced by radiation was investigated using synchronized populations of C3H10T1/2 cells. Mitotic-cell suspensions, collected using a mitotic shake-off procedure, were irradiated with 4Gy 250 kVp X-rays or 0.5 Gy fission neutrons from the RSV-TAPIRO reactor at CR-Casaccia. For study of cell killing the mitotic-cell suspensions were either irradiated immediately after collection, or plated for subsequent irradiation, which was performed every hour, covering an interval of 17 h. The response pattern observed was similar after X-rays and neutron irradiation, but the magnitude of the variation through the cell cycle was smaller in the case of neutrons (1.3- compared with 5-fold). For study of neoplastic transformation induction the irradiation was performed immediately after collection, i.e. in M phase, or at later times corresponding to mid-G1, G1/S and G2 phases. The sensitivity of the G2/M phase was examined by irradiating the cells with 4Gy X-rays while still attached to the flask bottom, and dislodging them after 25 min. SimilarLy to cell survival, the transformation frequency showed a small variation after neutron irradiation (1.4- compared with 3.1-fold) for the phases examined.

Somatic cell hybrids for high-density mapping of chromosome 2 breakpoints in radiation-induced myeloid leukemia cell lines from inbred mice†

Chromosome 2 (chr 2) deletions are recurrent abnormalities in acute myeloid leukemia (AML) induced by ionizing radiation in the mouse. The localization of deletion sites has proven extremely useful in providing information on the molecular mechanisms of leukemogenesis. The models available for the study of AML are mostly represented by inbred mouse strains, in which the molecular resolution of breakpoints is problematic. In this study, we have examined five leukemic cell lines exhibiting hemizygous chr 2 loss, derived from CBA, C3H, or (C57BL×CBA/H) F1 mice in which AML had been induced by a whole‐body dose of radiation. By application of a somatic cell hybridization technique, we have generated interspecific cell hybrids retaining the deleted murine chr 2 homologue. This strategy permitted a very detailed genetic analysis allowing the utilization of any genetic marker on chr 2 without a requirement for polymorphism. Somatic cell hybrid clones were subjected to a high‐density polymerase chain reaction–based microsatellite screening using 62–106 informative markers for each cell line. Detailed maps accurately defining chr 2 breakpoints were obtained. The identification of critical breakpoint markers allowed the construction of partial yeast artificial chromosome contigs across chr 2 breakpoints. These maps represent an essential resource for cloning of the breakpoint regions. Mol. Carcinog. 27:219–228, 2000. Published 2000 Wiley‐Liss, Inc.

Absence of a Dose-Fractionation Effect on Neoplastic Transformation Induced by Fission-Spectrum Neutrons in C3H 10T1/2 Cells

We have investigated the effect of fission-spectrum neutron dose fractionation on neoplastic transformation of exponentially growing C3H 10T1/2 cells. Total doses of 10.8, 27, 54, and 108 cGy were given in single doses or in five equal fractions delivered at 24-h intervals in the biological channel of the RSV-TAPIRO reactor at CRE-Casaccia. Both cell inactivation and neoplastic transformation were more effectively induced by fission neutrons than by 250-kVp X rays. No significant effect on cell survival or neoplastic transformation was observed with split doses compared to single doses of fission-spectrum neutrons. Neutron RBE values relative to X rays determined from data for survival and neoplastic transformation were comparable.

Neutron-induced tumors in BC3F1 mice: effects of dose fractionation.

An experimental study of the biological effectiveness of multifractionated low doses of high-LET radiation was carried out using BC3F1 male mice. They were treated with whole-body irradiation with five equal daily fractions of fission neutrons to yield cumulative doses of 0.025, 0.05, 0.10, 0.17, 0.25, 0.36, 0.535 and 0.71 Gy at the RSV-TAPIRO reactor (mean neutron energy 0.4 MeV, in terms of kerma, y D = 51.5 keV/microns, dose rate 0.004 Gy/min) and were followed for their entire life span. The statistical method described by Peto et al. (IARC Monograph, Suppl. 2, 1980) to establish the existence of a carcinogenic effect in long-term animal experiments was applied to the data sets. This analysis was done for myeloid leukemia and for the presence of selected solid tumors. Myeloid leukemia was absent in the control group and was rarely found in irradiated animals. However, a positive significant trend was found in the dose ranges 0-0.17 Gy and higher. Epithelial tumors were induced at doses from 0.17 Gy on. Tumor occurrence was evaluated further as final incidences with age adjustment for the differences in mortality rates. Survival and incidence data for selected classes of tumors after 0.17, 0.36 and 0.71 Gy were compared with those from a previous experiment at corresponding doses given acutely (dose rate between 0.05 and 0.25 Gy/min). This indicated no marked overall influence of the time regimen of neutron irradiation on survival and tumor induction.

Neutron Carcinogenesis in Mice: A Study of the Dose-Response Curves

Several experimental studies have been conducted with the objective to improve our knowledge of the types of dose-response relationships for radiation carcinogenesis in mice exposed to single acute doses. The experimental results on tumor induction have already been published and are here summarized with emphasis on the dependence on radiation quality, age at irradiation, and sex. These data indicate that the bone marrow, liver, and ovaries of the mice tested have an appreciable susceptibility to radiation carcinogenesis. However, the shape of the dose-response relationship depends on the tissue exposed. The data also confirm that a linear relationship is adequate for a conservative description of the dose-effect curves after exposure to low dose of neutrons, while a purely quadratic dependence is not inconsistent with the experimental data obtained using low-LET radiation. Other information which stems from the present analysis is that the susceptibility to radiation induction of liver tumor by fission neutrons decreases in old age. Finally, the experimental data on induction of ovarian tumors suggest a threshold-like dose response.